Uncovering the characteristics of the gut microbiota in patients with acute ischemic stroke and phlegm-heat syndrome

Growing evidence has indicated that the characteristics of gut microbiota are associated with acute ischemic stroke (AIS). Phlegm-heat syndrome (PHS), a specific pathological state of the AIS, is one of the common traditional Chinese syndromes of stroke. The long duration of PHS in patients with AIS could lead to poor clinical outcomes. Gut microbiota characteristics in patients with both AIS and PHS, and their relationship remains unknown. This study was designed to investigate the alterations in gut microbiota in patients with AIS and PHS through a cross-sectional study. Fecal samples were collected from 10 patients with AIS and non-PHS (ntAIS), 7 patients with AIS and PHS (tAIS), and 10 healthy controls (HC). Samples were profiled via Illumina sequencing of the 16S rRNA V3-V4. Stroke severity was assessed at admission by the National Institutes of Health Stroke Scale (NIHSS) and modified Rankin scale (mRS); their correlation with gut microbiota was investigated. The alpha-diversity of the bacterial communities was significantly higher in the fecal samples of patients with tAIS than in patients with ntAIS (Shannon index, P = 0.037). In addition, the combined tAIS and ntAIS group (tntAIS) exhibited higher microbiotic diversity when compared with HC (chao1, P = 0.019). The structure of intestinal microbiota was effectively distinguished between the tAIS and ntAIS group (ANOSIM, r = 0.337, P = 0.007). Additionally, the gut microbiota structure was significantly different between the tntAIS and HC groups (ANOSIM, r = 0.217, P = 0.005). The genera, Ruminococcaceae_ UCG_002 and Christensenellaceae_R-7_group, were implicated in the discrimination of PHS from non-PHS. The order Lactobacillales and family Lachnospiraceae were significantly negatively correlated with NIHSS and mRS at admission (P < 0.05). By contrast, the order Desulfovibrionales, families Christensenellaceae and Desulfovibrionaceae, and genera Ruminococcaceae UCG-014 and Ruminococcaceae UCG-002 were significantly positively correlated with NIHSS and mRS at admission (P < 0.05). This study is the first to profile the characteristics of gut microbiota in patients with AIS and PHS, compared with those with non-PHS. The genera, Ruminococcaceae_ UCG_002 and Christensenellaceae_R-7_group, may be objective indicators of this traditional Chinese medicine (TCM) syndrome in AIS. Furthermore, it provides a microbe-inspired biological basis for TCM syndrome differentiation.

hypertension, diabetes mellitus and hyperlipidemia, can lead to stroke. It is because of the influence of pretty confounding variable of cardiovascular risk factors among groups that we included the healthy controls with hypertension, diabetes mellitus or hyperlipidemia to exclude the influence of specific comorbidities and the baseline characteristics of the three groups were consistent and comparable (Table 1; all P > 0.05). We were very sorry that our inappropriate description caused your confusion.
The revised text reads as follows on Page 6, line 138: The HC group was defined as volunteers absent from parenchymal lesions of the major organs and past medical history of cardio-cerebrovascular disease through physical examination.
The baseline characteristics of the three groups were consistent and comparable, including age, sex, the body mass index, and comorbidities in order to exclude the influence of specific comorbidities (hypertension, diabetes, and hyperlipidemia) and other confounders that affect gut microbiota (Table 1; all P > 0.05).  Q3.The significance of the variations in microbial population in these diseased conditions need to be appropriately commented on.
Author response: We appreciated your valuable suggestions very much. We added the significance of the variations in microbial population into sections discussion to further elaborate the significance of the variations in microbial population.
The revised text reads as follows on Page 18, line 480.
This was the first study to explore the characteristics of the gut microbiota in AIS patients with PHS.

Ruminococcaceae_UCG_002 and
Christensenellaceae_R-7_group could be applied to determine and differentiate the profile of PHS, was a key finding of this study and provided novel insight for a reasonable explanation on the cause of constipation and more severe neurological dysfunction in the AIS patients with PHS,which suggested that early diagnosis and early intervention was of significant implication in clinical practice. Additionally, this study also suggested that supplementation of the diet with fiber, butyrate, or probiotics containing butyrate-producing bacteria could exert the neuroprotective effect on stroke recovery via promotion of consolidate epithelial integrity.

Reviewer 1
Author response: Thank you so much for your careful comment and review, which helped to revise and improve the manuscript. We hope the manuscript, after careful revisions, meets your high standards. The authors welcome further comments if any. Below we provide the point-by-point responses.
Q1. Line 37: Adding one line to explain the connection between AIS and PHS would be helpful.
Author response: Thank you so much for your advice, which we have now modified.
The revised text reads as follows on Page 2, line 24: Phlegm-heat syndrome (PHS), a specific pathological state of the AIS, is one of the common traditional Chinese syndromes of stroke.

Q2. Line 76: affected
Author response: We appreciated your considerate suggestions very much.
We have removed the spaces between the letters.
Author response: Thank you for your valuable advice. Considering the influence of pretty confounding variable of cardiovascular risk factors among groups, the healthy controls we included were relatively healthy with hypertension, diabetes mellitus or hyperlipidemia in order to exclude the influence of specific comorbidities. Briefly, the healthy controls were the populations with non AIS. We have added the definition of the healthy controls as you suggested.
The revised text reads as follows on Page 6, line 138: The HC group was defined as volunteers absent from parenchymal lesions of the major organs and past medical history of cardio-cerebrovascular disease through physical examination.
Q4. Line 114: The absence of cardiovascular risk factors in the healthy control group makes them a pretty confounding variable compared to the other two groups, whereby conclusions about differences between HC and either of the other groups has to really be taken with a lot of salt. Perhaps it would be prudent to further elucidate why the decision was made to exclude non-stroke patients, who had hypertension/diabetes/hyperlipidemia. The revised text reads as follows on Page 6, line 138: The HC group was defined as volunteers absent from parenchymal lesions of the major organs and past medical history of cardio-cerebrovascular disease through physical examination.
The baseline characteristics of the three groups were consistent and comparable, including age, sex, the body mass index, and comorbidities in order to exclude the influence of specific comorbidities (hypertension, diabetes, and hyperlipidemia) and other confounders that affect gut microbiota ( between chao1 and tntais. This is a serious flaw in the study design that needs to be addressed in terms of the logic behind combining the tAIS and ntAIS as well as the decision to exclude the confounds in the HC group. Author response: Thank you for pointing this out. Honestly, we wanted to drawed the conclusion of this study based on two key comparisons. First, the comparison between tAIS and ntAIS group contributed to the influence of PHS on the alterations of alpha-diversity. Second, we combined the tAIS and ntAIS group into a single tntAIS group, which were the patients with acute ischemic stroke. By comparison between tntAIS and HC groups, we could found the influence of acute ischemic stroke on gut microbiota. Species richness could be estimated with the Chao1 and shannon estimator. As shown in table 2, the shannon index were higher in tAIS group than those in ntAIS group with statistically significant difference. Additionally, the chao1 index were significantly higher in tntAIS group(acute ischemic stroke patients) than those in HC group (Table 3). Therefore, it could be concluded that acute ischemic stroke may result in the increased species richness of gut microbita, especially in AIS patients with PHS. As for the influence of pretty confounding variable of cardiovascular risk factors among groups, we included the healthy controls with hypertension, diabetes mellitus or hyperlipidemia to exclude the influence of specific comorbidities and the baseline characteristics of the three groups were consistent and comparable (Table 1; all P > 0.05). In order to avoid the confusion, we modified the manuscript as you suggested.
The revised reads as follow on Page 14, line 357.
The shannon index were higher in tAIS group than those in ntAIS group with statistically significant difference. Additionally, the chao1 index were significantly higher in tntAIS group(patients with AIS) than those in HC group.
Therefore, it could be concluded that AIS may result in the increased species richness of gut microbita, especially in AIS patients with PHS. The results of multiple diversities used to analyze the alpha and beta diversity of the community indicated that the alterations in richness and structure of gut microbiota existed in patients with AIS and more prevalent in those patients who also showed signs of PHS.
Q7. language, speech and neglect. Each impairment is scored on an ordinal scale ranging from 0 to 2, 0 to 3, or 0 to 4. Item scores are summed to a total score ranging from 0 to 42 (the higher the score, the more severe the stroke)[2-3].
Additionally, NIHSS is the most commonly used efficacy outcome for quantification of neurologic deficits in stroke-related meta analyses and was calculated as continuous variable and expressed as mean and standard deviations. Acute minor stroke in CHANCE study [4] was defined by a score of 3 or less at the time of randomization on the NIHSS. Therefore, stroke severity at admission in our study was assessed using the NIHSS.
[ Author response: We appreciate your considerate suggestions very much.
We have modified the sentences as you suggested.
The revised text reads as follows on Page 11, line 225: The relative abundance of Firmicutes and Bacteroidetes in the ntAIS and tAIS group showed a trend of lower values than that of the HC group. By contrast, the relative abundance of Actinobacteria and Proteobacteria in the ntAIS and tAIS group showed a trend of higher values than that of the HC group.
However, they failed to reach the level of statistical significance.
Q3. The manuscript is written in acceptable English. There is no noticeable grammar/syntax/spelling mistakes. However, there are a few typos.
Line 276: typo in the reference citation Line 297: typo in the reference citation Author response: We were really sorry for our careless mistakes and have corrected all these errors.
Q5. In the text body, Figure 3A is mentioned before Figure 2B. The text would flow better if figures were in order. If possible, please adjust the text or figure order. If not, kindly explain.
Author response: We appreciated your considerate suggestions very much.
We have modified the figure order as you suggested.
Q6. Figure 2: Mention in the legend how the abundance was calculated.
Author response: Thanks for your considerate suggestions very much. We have revised the figure lengend as you suggested.
The relative species abundances were calculated as percentages of the total species abundances(the number of tags measured in the sample divided by the total number of tags). Author response: Thank you for pointing this out. We have revised the legend of figure 3 as you suggested. We are sorry that the figures that you have seen were of poor quality. As such, we have enhanced the resolution of all figures and uploaded them again. Figure 4: Mention what a low or high LDA score means.

4)
Author response: Thank you so much for your valuable advice. We have modified the legend of Figure 4 as you suggested.
The LDA score was obtained by LDA (linear regression analysis); the larger the LDA score, the greater the influence of species abundance on the difference effect.